1. Field of the Invention
Soybean rust is caused by two species of fungi, Phakopsora pachyrhizi Sydow and P. meibomiae (Arthur) Arthur. This invention relates to novel PCR primers and the development of both classical and real-time PCR assays for the rapid detection and discrimination of the soybean rust pathogens P. pachyrhizi and P. meibomiae. 
2. Description of the Relevant Art
Soybean rust is a devastating disease in several soybean growing regions of Asia, Australia, and Africa, and is a potential threat to other countries where soybeans are grown. Soybean rust has been reported in China, Taiwan, Thailand, India, Japan, and Australia in the Eastern Hemisphere and in Brazil, Colombia, Costa Rica, and Puerto Rico in the Western Hemisphere (Asian Vegetable Research and Development Center. 1987. Bibliography of Soybean Rust, 1985–1986. AVRDC. Tainan, Taiwan. 103 pages). Yield losses of up to 70–80% have been reported in some fields in Taiwan (Bonde et al. 1976. Phytopath. 66:1290–1294; Bromfield, K. R. 1984. Soybean Rust, Monograph No. 11. APS Press Inc., St. Paul, Minn., 65 pages). Plants that are heavily infected have fewer pods and smaller seeds that are of poor quality (Bromfield, supra). While soybean rust was found in Hawaii in 1994, it has not yet been observed in the continental U.S. (Sinclair et al. 1996. Soybean Rust Workshop, Aug. 9–11, 1995. College of Agricultural, Consumer, and Environmental Sciences, National Soybean Research Laboratory Pub#1, Urbana, Ill., 850 pages).
Soybean rust is caused by two morphologically similar species of Phakopsora: Phakopsora pachyrhizi Sydow and P. meibomiae (Arthur) Arthur (Ono et al. 1992. Mycol. Res. 96: 825–850). P. pachyrhizi occurs throughout Australia, Asia, and the islands of Japan, The Philippines, and Taiwan (Ono et al., supra). The soybean rust pathogen recently reported in Hawaii (Sinclair et al., supra) and Zimbabwe (C. Levy, Personal communication) has been tentatively identified as P. pachyrhizi. P. meibomiae is found in South and Central America and the Caribbean (Ono et al., supra).
Soybean rust has been identified as a potentially devastating disease if the pathogen were to gain entry and become established in the U.S. Both P. pachyrhizi and P. meibomiae can infect an unusually broad range of plant species. P. pachyrhizi naturally infects 31 species in 17 genera of legumes, and 60 species in 26 other genera have been infected under controlled conditions (Sinclair et al., supra). P. meibomiae naturally infects 42 species in 19 genera of legumes, and 18 additional species in 12 other genera have been artificially infected. Twenty-four plant species in 19 genera are hosts for both species (Sinclair et al., supra).
Although both pathogens damage plants, P. pachyrhizi is more aggressive and causes considerably more yield loss (Sinclair et al., supra). Previously, isozyme analysis was successful in discriminating between these two Phakopsora species (Bonde et al. 1988. Phytopath. 78:1491–1494). However, this method is slow and is not useful for detecting and identifying the pathogens in infected plant material. Field identification of soybean rust often is difficult, because symptoms are easily confused with bacterial pustule caused by Xanthomonas axonopodis pv. glycines, especially during the early stages of disease development (1999. Compendium of Soybean Diseases, 4th Edition, Hartman et al., Eds. APS Press Inc., St. Paul, Minn., 100 pages; Sinclair et al., supra; Tschanz et al. 1985. In: Proc. World Soybean Research Conference III, R. Shibles, Ed. Westview Press, Boulder, Colo., pages 562–567). Even using a hand lens, the lesions of the two diseases on the upper leaf surface look very similar. Likewise, the raised dried blisters of the bacterial pustule lesions on the underside of the leaf appear similar to the uredinial cones of soybean rust (Sinclair et al., supra). Therefore, a molecular-based diagnostic assay that is specific to the soybean rust pathogens, like PCR, would be extremely helpful in making an accurate and timely identification.
The recent findings of soybean rust in Hawaii and Zimbabwe, and the re-emergence of the disease in India, has prompted fears that the pathogen(s) are spreading to new geographic regions. If P. pachyrhizi were to gain entry into the continental U.S. and become established, serious losses would likely occur (Yang et al. 1991. Plant Dis. 75: 976–982). It has been estimated that yield losses could exceed 10% in most of the U.S., and up to 50% in the Mississippi delta and southeastern states (Sinclair et al.; Yang et al., supra).
Currently, there is no resistance to soybean rust in any of the U.S. commercial soybean cultivars. Some fungicides have been found to be effective against P. pachyrhizi by slowing the spread of the pathogen enough so that normal seed set and pod fill can occur (Sinclair et al., supra). However, widespread fungicide applications on soybean fields in the U.S. are not deemed cost effective. As a result, this control option would be useful only for eradication on small acreages. Accurate and timely diagnoses of plant diseases are extremely important so that appropriate control measures and/or eradication procedures can be implemented quickly at an early stage of infection to slow the spread of the pathogen and reduce yield losses. Disease symptoms often aid with making decisions, but a definitive diagnosis requires unambiguous pathogen identification.
There exists a need for new technologies to be examined and novel methods to be developed for the detection and identification of exotic plant pathogens that are deemed significant threats to United States agriculture. Thus, specific primers and methods capable of specifically identifying and differentiating pathogenic P. pachyrhizi and P. meibomiae isolates are needed.